Cell or tissue cultivation apparatus and method of cultivation

ABSTRACT

A cultivation apparatus and a cultivation method of a culture such as a cell or tissue. Provided are an incubator unit for accommodating a culture and a lever that penetrates through the incubator unit and can move in circular arc around a fulcrum as a center, the fulcrum being set at a wall of the incubator unit or in the vicinity thereof, then displacement is imparted to the culture by operating the lever. Bending force can be acted on the culture (cell construct) such as a cell or tissue, so that without any increase or decrease of a culture fluid in the incubator unit, namely, without any increase or decrease of pressure to the culture fluid, displacement required for cultivation can be imparted to the culture in the incubator unit. By curving, continuous compression and extension are generated in a direction of thickness from a concave portion to a convex portion of the curving.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Phase filing under 35 U.S.C. §371 ofPCT/JP2007/062409 filed Jun. 20, 2007, which claims priority to PatentApplication No. 2006-189733, filed in Japan on Jul. 10, 2006. The entirecontents of each of the above-applications are incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to cultivation of a cell or tissue in thefields of regeneration medicine and tissue engineering, and relates to amethod of three dimensional tissue cultivation for three dimensionaltissue or organ regeneration. Concretely, the present invention relatesto a cultivation apparatus and a cultivation method that are used incultivation executed with one or some of a cell, a cell scaffold and anECM (extracellular matrix) that a cell generates, as a cell construct.There may be a case where the above cultivation is executed withaddition of a culture fluid, other additives, a growth factor, achemical and so on.

In short, the cultivation apparatus and the cultivation method of thepresent invention differ from the conventional static cultivation. Thecultivation apparatus and the cultivation method of the presentinvention are an apparatus relating to three dimensional cultivationwhere physical action is used together. The cultivation apparatus is forrealizing objective regeneration tissue by promotion of differentiationor stop thereof along with growth, cell migration and substancemigration that are promoted by stimulating a cell of a cell constructaggressively and displacing a cell construct.

2. Description of the Related Art

For cultivation of a cell or tissue, a method of imparting physicalstimulation such as pressure and tension to a cell or tissue to becultivated is studied, and various bioreactors and so on are suggested.Two dimension cultivation (plane cultivation) is a cultivation methodusing a flat bottomed culture carrier, and in general, is staticcultivation in an incubator. Suspension cultivation is a method ofcultivating a non-adherent cell being suspended. This method is alsostatic cultivation in an incubator. Three dimension cultivation is amethod that is generally executed such that a cell scaffold where a cellis disseminated is left still in an incubator to be cultivated. It isgeneral for the three dimension cultivation (using a bioreactor) that acell is made to adhere to or is enclosed by a cell scaffold to processstirring of a culture fluid and so on. It is conceived that in the threedimension cultivation of a cell scaffold, physical action such aspressure, compression, tension and shear are imparted to a cell.

A cultivation apparatus for imparting physical action is called “abioreactor”, “a tissue engineering processor”, etc. Such apparatus isexpected to be into practical utilization as a cell/tissue cultivationapparatus in vitro for cultivation experiments of tissue engineering andregeneration medicine.

Concerning such bioreactor having functions of cultivating a cell ortissue, and imparting physical displacement, stress and stimulation usedin the cultivation, a method for cultivating a cell or tissue and anapparatus therefor are disclosed in Japanese Laid-open PatentPublication No. 2001-238663 (Abstract, etc.) as an example of usingpressure and oscillation (supersonic wave), a method for in vivo, exvivo and in vitro, repair and regeneration of cartilage and collagen,and bone remodeling is disclosed in Published Japanese Translations ofPCT International Publication for Patent Application No. 2004-512031(Abstract, etc.) as an example of using pressure, a cell andtissue-cultivating apparatus is disclosed in Japanese Laid-open PatentPublication No. 2002-315566 (Abstract, etc.) as an example of usingshear force, a cell and tissue-cultivating device is disclosed inJapanese Laid-open Patent Publication No. 2003-061642 (Abstract, etc.)as an example of using tensile force, a cell and tissue cultivationapparatus is disclosed in Japanese Laid-open Patent Publication No.2003-180331 (Abstract, etc.) as an example of using compression force, adevice for cultivating cell is disclosed in Japanese Laid-open PatentPublication No. H09-313166 (Abstract, etc.) as an example of using shearforce, a loading device of extending and contracting stimulation forcultivating a cell by using a silicone belt is disclosed in JapaneseLaid-open Patent Publication No. H10-155475 (Abstract, etc.) as anexample of using tensile force, and an apparatus executingsterilization, inoculation, cultivation, preservation, transport andtest of tissue and a synthetic or natural vascular graft, and a methodtherefor are disclosed in Published Japanese Translations of PCTInternational Publication for Patent Application No. H11-504216(Abstract, etc.) as an example of using both tension and shear. Acultivation method where distortion is given to cells held on membranesby the membranes is disclosed in Japanese Laid-open Patent PublicationNo. 2005-143343 (Abstract, etc.). A semi-permeable membrane being usedfor cultivation is disclosed in International Publication Pamphlet No.WO 2006/015304 A2 (Abstract, etc.) and Published Japanese Translationsof PCT International Publication for Patent Application No. 2000-513214A (Abstract, etc.). Imparting of various kinds of physical action andstimulation, and using of a semi-permeable membrane are tried forcultivation of a cell, etc.

There are regions receiving many kinds of stress in the human body.Tissue used for repairing these regions is different according to theregions. For example, a disc, a meniscus, a bone, fiber cartilage and avalve of a heart receive bending force in vivo. This bending stress isdifferent from simple pressure, compression, tension, shear, etc. It isinsufficient that tissue cultivated by a stimulus factor such as asimple pressure, compression, tension and shear is applied to a regionreceiving such bending force.

For the above, the inventors of the present invention conceive thatbending is so useful for growth, etc. of a cell or tissue as stimulationor a load imparted to a cell or tissue to be cultivated. Such problem isnot disclosed in the above patent documents, and is not also suggestedtherein.

SUMMARY OF THE INVENTION

An object of the present invention relates to a cultivation apparatusand a cultivation method for a culture including a cell and/or tissue,and is to provide an apparatus and a method for cultivating a celland/or tissue proper for a region of a body of a human being and so on.

Another object of the present invention relates to a cultivationapparatus for a cell and/or tissue proper for a region of a body of ahuman being, etc., and to contribute cultivation of a proper cell and/ortissue by curving, or extending and compressing a culture.

To achieve the above object, the cultivation apparatus of the presentinvention can act bending force on a culture including a cell and/ortissue, so that without any increase or decrease of a culture fluid inan incubator unit, namely, without any increase or decrease of pressureon the culture fluid, displacement required for cultivation can beimparted to the culture in the incubator unit. Concretely, by curving,continuous compression and extension are generated in a direction ofthickness from a concave portion to a convex portion thereof. Byapplying to a culture physical stimulation or a load not attained byconventional pressurization, shear and tension, a culture appropriatefor restoration of tissue at a region accompanying bending is realized.The present invention is not limited to such bending, and without anyincrease or decrease of a culture fluid in an incubator unit, namely,without any increase or decrease of pressure on the culture fluid,motion, stimulation, etc. that are necessary for cultivation can beimparted to a culture.

To achieve the above object, a first aspect of the present inventionthere is provided a cultivation apparatus for a culture including a celland/or tissue, comprising an incubator unit that accommodates theculture; and a lever that penetrates from an inside of the incubatorunit to an outside thereof, wherein displacement is imparted to theculture by operating the lever. From such structure, the above objectscan be achieved.

To achieve the above object, a second aspect of the present inventionthere is provided a cultivation apparatus for a culture including a celland/or tissue, comprising a bed on which the culture is disposed; anincubator unit that accommodates the culture with the bed; a lever thatpenetrates from an inside of the incubator unit to an outside thereof;and a driving unit that pushes the bed by operating the lever, andimparts displacement to the culture by curving deformation of the bed.From such structure, the above objects can be achieved.

To achieve the above object, in the above cultivation apparatus,preferably, the lever may move in circular arc around a fulcrum as acenter, the fulcrum being set at a wall of the incubator unit or in thevicinity thereof. From such structure, the above objects can beachieved.

To achieve the above object, a third aspect of the present inventionthere is provided a cultivation apparatus for a culture including a celland/or tissue, comprising a bed on which the culture is disposed; anincubator unit that accommodates the culture with the bed; a lever thatpenetrates from an inside of the incubator unit to an outside thereof,and can move in circular arc around a fulcrum as a center, the fulcrumbeing set at a wall of the incubator unit or in the vicinity thereof;and a driving unit that imparts stretching force to the bed by operatingthe lever, and extends and contracts the culture by deformation due toextension and contraction of the bed. From such structure, the aboveobjects can be achieved.

To achieve the above object, a fourth aspect of the present inventionthere is provided a cultivation method for a culture including a celland/or tissue, comprising the steps of accommodating the culture in anincubator unit; imparting movement in circular arc around a fulcrum setat a wall of the incubator unit or in the vicinity thereof as a center,to a lever penetrating from an inside of the incubator unit to anoutside thereof; and imparting bending displacement to the culture bythe movement in circular arc. From such structure, the above objects canbe achieved.

According to the present invention, following effects can be obtained.

(1) Displacement (stress) such as bending is applied to a culture incultivation, and bending motion can be purely generated.

(2) It is able to be used for regeneration of tissue receiving bendingforce in vivo like discs, etc.

(3) It can be expected to prevent a stem cell from differentiating andprevent a tissue cell from dedifferentiating, and if tissue structureand so on have directionality, an arranging direction thereof can beuniform, and a culture equivalent to tissue in vivo can be obtained.

(4) A necessary tissue can be cultivated by bending action without otherkinds of physical action such as pressure, or with the minimum thereof.

(5) Cell migration can be expected to become easy.

(6) Nutrients and oxygen can be osmosed in the interior of a threedimensional cell construct, and discharging waste products is expected.

The object and advantages of the invention will be realized and attainedby means of the elements and combinations particularly pointed out inthe claims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory and are not restrictive of the invention, as claimed.

Other objects, features and advantages of the present invention are moreclearly understood by referring to attached drawings and each ofembodiments.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a vertical sectional view showing a culture unit of acultivation apparatus according to a first embodiment;

FIG. 2 is an exploded perspective view showing an incubator unit;

FIG. 3 is an exploded perspective view showing of an incubator bodyincluding a cover unit;

FIG. 4 is a perspective view showing a bottom surface of a cover unit;

FIG. 5 is an exploded perspective view showing a driving unit;

FIG. 6 is a plan view showing a culture unit in a state where a coverunit is taken off from an incubator unit;

FIG. 7 is a perspective view showing an enlarged culture bed;

FIG. 8 is a view showing an actuator;

FIG. 9 is a plan view showing an actuator;

FIG. 10 is a view showing one example of a movement converting unit inan actuator;

FIG. 11 is a view showing one example of motion of a movement convertingunit;

FIG. 12 is a block diagram showing one example of a control system of acultivation apparatus;

FIG. 13 is a flowchart showing processing procedure of cultivation;

FIG. 14A is a view showing a culture bed where a cell construct isdisposed;

FIG. 14B is a view showing a culture bed where a cell construct isdisposed;

FIG. 15A is a view showing bending motion to a cell construct;

FIG. 15B is a view showing bending motion to a cell construct;

FIG. 16 is a flowchart showing operation procedure of an actuator;

FIG. 17 is a view showing a structural example of a cell constructaccording to a second embodiment;

FIG. 18 is a view showing another structural example of a cellconstruct;

FIG. 19 is a view showing another structural example of a cellconstruct;

FIG. 20 is a view showing a cultivation system according to a thirdembodiment;

FIG. 21 is a block diagram showing a control system of a cultivationsystem;

FIG. 22 is a flowchart showing processing procedure of a cultivationsystem;

FIG. 23 is a view showing a culture unit of a cultivation apparatusaccording to a fourth embodiment;

FIG. 24 is a sectional view of FIG. 23 along an XXIV-XXIV line;

FIG. 25 is a view showing an experimental example;

FIG. 26 is a view showing an experimental example;

FIG. 27 is a view showing an experimental example;

FIG. 28 is a view showing an experimental example; and

FIG. 29 is a view showing an experimental example.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS First Embodiment

A first embodiment of the present invention will be described withreferring to FIGS. 1 to 12. FIG. 1 is a vertical sectional view showinga culture unit according to a first embodiment of a cultivationapparatus, FIG. 2 is an exploded perspective view showing an incubatorunit, FIG. 3 is an exploded perspective view showing of an incubatorbody including a cover unit, FIG. 4 is a perspective view showing abottom surface of a cover unit, FIG. 5 is an exploded perspective viewshowing a driving unit, FIG. 6 is a plan view showing a culture unit ina state where a cover unit is taken off from an incubator unit, FIG. 7is a perspective view showing an enlarged culture bed, FIG. 8 is anelevation view showing an actuator, FIG. 9 is a plan view showing anactuator, FIGS. 10 and 11 depict a concrete example of a movementconverting unit in an actuator, and FIG. 12 depicts one example of acontrol system of a cultivation apparatus. In FIGS. 10 and 11, the samecomponents as an actuator of FIGS. 8 and 9 are denoted by the samereference numerals.

This first embodiment shows a culture unit that imparts bendingdisplacement to a cell construct which is a culture. This culture unit 2includes, as shown in FIG. 1, an incubator unit 4 as a culture chamberand a driving unit 8 that imparts desired movement to a culture in theincubator unit 4, for example, a cell construct 6. Structure and afunction of each function unit will be described, and a cultivationmethod will be referred as follows.

A) Incubator Unit (Culture Chamber) 4

The incubator unit 4 is a culture space for the cell construct 6, and isa function unit of imparting a culture fluid 48 (FIG. 6), anddisplacement or stimulation that is necessary for cultivation to thecell construct 6. Then, this incubator unit 4 includes, as shown in FIG.2, an incubator body 10, an incubator bottom 12 and a cover unit 14. Theincubator body 10 is, for example, a flat barrel, and forms acylindrical culture space 16.

The incubator bottom 12 includes a diaphragm 20 that closes the culturespace 16, and is attached to a bottom surface of the incubator body 10by a plurality of fixing screws 23 through the medium of a sealingmeans, for example, an O-ring 22. The diaphragm 20 is disposed forapplying pressure to the culture space 16 from an outside. Abarrel-shaped standing wall 24 is formed on a part of the incubatorbottom 12, the part corresponding to the culture space 16. A window 26enclosed by the standing wall 24 is formed. A pressure sensor 29 isdisposed along with a pressure apparatus 27. The pressure apparatus 27applies pressure P to the culture space 16 via the diaphragm 20 by themedium of a fluid such as water. This pressure P is detected by thepressure sensor 29.

On the top of the incubator body 10, as shown in FIG. 3, a window 30 isformed by a barrel-shaped standing wall 28. O-rings 32 and 34 areintervened inside and outside the standing wall 28 respectively, andthereby, the cover unit 14 is attachably and detachably installed to theincubator body 10.

In an inner wall surface of the culture space 16 of the incubator body10, shelves 38 and 40 are formed. A culture bed 36 is disposed over bothof the shelves 38 and 40. In the incubator body 10, positioningprotrusions 42 and 44 (FIG. 6) are formed for the culture bed 36. Anedge of the culture bed 36 is touched to one positioning protrusion 42,and the other positioning protrusion 44 is engaged with an engagingconcave 46 of the culture bed 36. Thereby, the culture bed 36 ispositioned at a predetermined position in the culture space 16, and isheld attachably and detachably. That is, a position in height of theculture bed 36 is determined by the selves 38 and 40. A position in ahorizontal direction and an angle thereof are determined by positions ofthe positioning protrusion 42 and the culture bed 36, positionalrelationship between the positioning protrusion 44 and the engagingconcave 46, and engagement therewith.

The culture bed 36 is also held in a vertical direction of the culturespace 16. As shown in FIG. 4, on a bottom face of the cover unit 14, aplurality of supporting protrusions 47 that protrude to the culture bed36 are formed. These supporting protrusions 47 contact an edge of theculture bed 36, and hold the culture bed 36. Thus, free movement of theculture bed 36 in a vertical direction is prevented.

In the incubator body 10, as shown in FIG. 6, an inlet port 50 that runsin the culture fluid 48 to the culture space 16, and an outlet port 52that exhausts the culture fluid 48 from the culture space 16 are formed.A penetration hole 54 in the inlet port 50 is opened at the bottom ofthe shelf 38 that the culture bed 36 is disposed, and communicates witha through hole 56 that opens in the positioning protrusion 42. Apenetration hole 58 in the outlet port 52 is opened at the bottom of theshelf 40 that the culture bed 36 is disposed, and communicates with athrough hole 60 that opens in the shelf 40. Therefore, the culture fluid48 supplied from the inlet port 50 runs under the shelf 38, runs overthe culture bed 36 from the through hole 56 of the positioningprotrusion 42, and reaches the penetration hole 58 via an inside of theculture space 16. A part of the culture fluid 48 reaches the outlet port52 from the top of the culture bed 36 via the through hole 60 of theshelf 40. A circulation pipe 51 is connected to the inlet port 50, and acirculation pipe 53 is connected to the outlet port 52. Thesecirculation pipes 51 and 53 compose a circulation path for the culturefluid 48.

To the incubator body 10, as shown in FIGS. 1, 2, 3 and 6, a joint part62 is fixed by attachment screws 65. Between the culture space 16 andthe driving unit 8, a lever 70 is disposed across the wall of theincubator body 10. This lever 70 can rotate in a circular arc around afulcrum 72 (FIG. 1) as a center, the fulcrum 72 being set at the wall ofthe incubator body 10. By driving force imparted by the driving unit 8,a tip of the lever 70 moves up and down. By contacting the tip of thelever 70 on the culture bed 36, the culture bed 36 can be curved, anddisplacement motion can be imparted to the cell construct 6 that is heldby the culture bed 36.

The lever 70 includes, as shown in FIG. 3, an action part 74, a smallerdiameter part 76, a larger diameter part (sealing part) 78, a flange 80,a cylinder 82 and a cone 84 whose tip is the sharpest part in the lever70 in order from the culture space 16 side. The flange 80 has a flatface at a front side when seeing from the action part 74 side, and has asliding face 86 formed by a sphere at a rear side.

A penetration hole 88 for penetrating the lever 70 is formed in theincubator body 10. The penetration hole 88 is formed in a largerdiameter at the culture space 16 side than that at an opposite side forallowing the lever 70 at the culture space 16 to rotate. In order tomaintain airtightness between the lever 70 and the wall of the incubatorbody 10, an O-ring 90 is provided. The center of the O-ring 90 is thecenter of rotating of the lever 70, and constitutes the above describedfulcrum 72. Between the O-ring 90 and the flange 80 of the lever 70, anannular lever guide 92 is disposed. The lever 70 is constituted torotating around the center of the O-ring 90 as a center of rotating.

The joint part 62 is a member for connecting a frame 64 of the drivingunit 8 to the incubator body 10. As shown in FIG. 6, the joint part 62is fixed by attachment screws 66 and 68. A projection 96 (FIG. 3) of thejoint part 62 is engaged with a concave 98 (FIG. 1) corresponding to thepenetration hole 88. On the projection 96, a sliding face 100 thatcontacts the sliding face 86 of the flange 80 of the lever 70 and isformed by a sphere is formed. In the joint part 62, a through hole 102that has a larger diameter at an opening side than that at an oppositeside for allowing the lever 70 to rotate is formed.

In the incubator unit 4, the diaphragm 20 is, for example, formed by aflexible material such as silicone rubber that does not liquate out andis high safety. The diaphragm 20 is formed into a thin shape, and theO-ring 22 is formed therearound. The reason why the diaphragm 20 is athin membrane is for transmitting outside pressure P into the culturespace 16, and for making pressure in the incubator unit 4 almost thesame pressure as pressure in an area beyond the diaphragm 20. The lever70 is sealed by the O-ring 90, then airtightness and water-tightness ofthe incubator body 10 are maintained.

B) Driving Unit 8

The driving unit 8 is a function unit that receives driving forceimparted from an outside, and drives the lever 70 imparting displacementmotion to the cell construct 6. To the frame 64 of the driving unit 8,as shown in FIGS. 1 and 5, the cover part 104 is fixed by a plurality ofscrews 106. The cover part 104 receives driving force via a wire 110. Tothe cover part 104, a first slider 112 that moves up and down isslidably attached. A tip of the slider 112 touches on the lever 70. Inthe frame 64, a second slider 114 that is disposed on the same line asthe slider 112 is provided. Between the slider 114 and the frame 64,provided is a spring 116 that acts restoring force on the slider 114 tomove the slider 114 upward. The spring 116 composes a restoring springthat replaces the slider 112 to a position before movement.

The wire 110 is attached at its tip to the slider 112. An outer tube 118is provided around an outside face of the wire 110. The wire 110 isslidable independently from the outer tube 118. A cable 119 is composedof the wire 110 and the outer tube 118. The outer tube 118 penetrates afixing nut 120, and is fixed to the cover part 104 by the fixing nut120. Between the fixing nut 120 and the cover part 104, a sliding bush122 is disposed. An actuator 124 (FIGS. 8 and 9) is attached to an edgeof the wire 110 as a driving source for moving the slider 112 up anddown. In this case, if the slider 112 falls, the slider 114 is presseddown along with the lever 70, and if driving force of the actuator 124is released, the sliders 112 and 114 restore to original positions byrestoring force of the spring 116.

In the frame 64, an opening 126 for inserting the lever 70 is formed.Guide plates 128 and 130 that guide insertion of the lever 70 areinserted between the sliders 112 and 114 from the opening 126. If thecone 84 of the lever 70 is inserted between the guide plates 128 and130, the lever 70 can be easily inserted into a predetermined positionbetween the sliders 112 and 114.

C) Culture Bed 36

The culture bed 36 is a means for holding the cell construct 6 and fortransmitting displacement motion to the cell construct 6, and is afunction unit returning the cell construct 6 to a state beforedisplacement motion by using elasticity that the culture bed 36 has. Theculture space 16, for example, accommodates the cell construct 6disposed on the culture bed 36.

A disposing part 136 where two cell constructs 6 are disposed inparallel is included in the culture bed 36 as shown in FIG. 7. Thisdisposing part 136 constitutes a receiving part that is deformed byreceiving action from an outside, has an area and a shape for disposingtwo cell constructs 6 in parallel, and is made in a plate form from anelastic member for imparting bending motion to each cell construct 6. Asan elastic member, for example, a stainless steel sheet for a spring orother materials that have high spring are used. In this case, the wholeculture bed 36 may be formed by an elastic member, or the disposing part136 that enables bending motion or a part of the disposing part 136 maybe formed by an elastic member. The deposing member 136 is not limitedto a flat-plated shape, and may be net. Stable coating may cover thedisposing part 136 to prevent component of a spring member fromliquating out.

The disposing part 136 is a rectangular shape. At end parts in a longerdirection thereof, rectangular standing walls 138 and 140 are formed.Each of the standing walls 138 and 140 is perpendicular to the disposingpart 136, and in the standing walls 138 and 140, elliptic through holes142 where each cell construct 6 is inserted are formed. These throughholes 142 have a role of fixing both ends of the cell construct 6. Eachof the standing walls 138 and 140 is set in a predetermined height haccording to a size of each cell construct 6.

At a top of each of the standing walls 138 and 140, supporting faces 144and 146 that have a constant width in parallel to the disposing part 136and are formed. From each supporting face 144 and 146, a turnover 148 isformed in parallel to each of the standing walls 138 and 140 by turninga part of each supporting face 144 and 146. Each turnover 148 reinforceseach supporting face 144 and 146, and each standing wall 138 and 140,and fixes the cell construct 6. That is, sufficient strength can beobtained if each supporting face 144 and 146, and each standing wall 138and 140 are formed by the same board as the disposing part 136 which ismade of a thin plate, and, in the embodiment, the culture bed 36 is bentonly in a longitudinal direction. In the culture bed 36, the U-formedengaging concave 46 corresponding to the positioning protrusion 44 asshown in FIG. 6 is formed in order to fix the supporting face 144.

From middle edges of the disposing part 136, supporting walls 152 and154 that support sides of the disposed cell construct 6 are formed. Froma top of each supporting wall 152 and 154, holding parts 156 and 158that cover a top surface of the cell construct 6 are formed. Eachsupporting wall 152 and 154 is a wall perpendicular to the disposingpart 136. The height thereof is the same as the above described standingwalls 138 and 140. Each holding part 156 and 158 constructs a parallelface with the disposing part 136. The cell construct 6 is disposed in agap between the disposing part 136, and each holding part 156 and 158.An end part of each holding part 156 and 158 constructs a curve face.Between the end parts, a gap 160 for attaching and detaching the cellconstruct 6 is set.

Concerning the incubator unit 4, components thereof are selected in viewof stability, economics, sterilizationproof, workability, abrasionresistance, handleability, etc. for a culture and a culture fluid. Forcomponents of contacting the culture fluid 48, for example, theincubator body 10, the cover unit 14, the O-rings 32, 34 and 90, theculture bed 36 and the lever 70, a material of high stability that doesnot liquate out must be used. For example, stainless steel, plastic,etc. can be used. Stainless steel is superior in stability andsterilizationproof, and plastic is superior in workability andhandleability such as weight saving and disposability.

If plastic is used, disposable use can be performed for a need ofpreventing pollution by germs and pollution by a cell or a gene ofanother person. Plastic is cheaper than stainless steel. The incubatorunit 4 must be sterilized in an assembled state. Both stainless steeland plastic are proof against damage from a sterilization process. Forexample, full heatproof characteristic is requested in an autoclave thatexecutes a sterilization process on condition of 121° C. and 2 pressure.For such process, fluororesin such as PTFE, ETFE and PFA, polysulfane,polyethersulfane, polycarbonate, PET and polyethersulfane reinforced byglass, etc. are suitable. For a sterilization process by a γ-ray or anelectronic ray, ETFE, polysulfane, polyethersulfane, polycarbonate, PET,polyethylene, polypropylene, etc. are suitable.

In the incubator unit 4, abrasion resistance is requested becausesliding of the flange 80 of the lever 70 is repeated at the joint part62 and the lever guide 92, etc. Then, fluororesin such as polyacetal,polyethylene, polypropylene, PTFE, ETFE, and PFA is suitable.

For observing an inside of the cover unit 14 without taking off thecover unit 14, a transparent material is suitable. For the cover unit14, a transparent material such as polysulfane, polyethersulfane,polycarbonate, PET, etc. are suitable.

In order to prevent pollution of a culture, a sterilization process isnecessary. Selection of a material in view of proof to sterilization isimportant. The best materials that endure a sterilization by both of anautoclave and a γ-ray or an electronic ray are as follows in view offunctionality, etc. that each component needs.

For the incubator body 10, the incubator bottom 12 and the cover unit14, polysulfane, polyethersulfane or polycarbonate is suitable. For theO-rings 32, 34 and 90, fluoro-rubber or silicone rubber is suitable. Forthe diaphragm 20 and the O-ring 22, fluoro-rubber, silicone rubber or anETFEE film is suitable. For the joint part 62 and the lever guide 92,ETFE is suitable. For the lever 70, stainless steel (SUS316 and SUS304)is suitable. For the culture bed 36, stainless steel for a spring (aseries of SUS304CSP) is suitable.

Stainless steel for a spring is less corrosion resistance than a seriesof SUS316. For supplement this less corrosion resistance, for example,coating such as diamond-like coating (DLC) may be covered.

If reusing is expected with washing carefully, instead of plastic,stainless steel SUS316 or SUS304 may be used. As to a component materialof the culture bed 36, a function of occurring displacement movement byapplying pressure and returning to a state before displacement isnecessary. So, for example, a hard elasticity member such as stainlesssteel for a spring may be used.

D) Actuator 124

The actuator 124 constitutes a driving source that imparts driving forceof the lever 70 from an outside to the driving unit 8. As shown in FIGS.8 and 9, the actuator 124 converts rotation of a motor to back and forthmove of the wire 110, and applies move thereof to the slider 112 of thedriving unit 8. The wire 110 of the driving unit 8 is guided to theactuator 124 along with the outer tube 118, and the tip of the wire 110is fixed to a crank lever 162 by a wire fixing screw 164. The outer tube118 that covers the wire 110 is fixed to a fixing part 170 of areinforcing plate 168 that is disposed on the frame 166 of the actuator124 as a relay member.

The crank lever 162 is fixed to a crank shaft 174 via a baring 172. Amotor 178 is attached to a crank 176 where the crank shaft 174 is fixed.The motor 178 is, for example, constituted of a DC motor. With thismotor 178, an encoder 180 that detects rotation is disposed.

A rotation adjuster 182, a power source switch 183, an operation switch184, a rotation adjusting dial 186 and a display 188 that displaysnumber of rotations, etc. are disposed on the frame 166 of the actuator124.

As an example showing an enlarged crank mechanism of this actuator 124,as shown in FIG. 10, a crank arm 192 showing the crank 176 is attachedto a rotation shaft 190 of the motor 178. To the crank arm 192, thecrank lever 162 is rotatably attached by the crank shaft 174. To thecrank lever 162, the tip of the wire 110 is fixed by the wire fixingscrew 164.

According to such structure, by rotation of the motor 178, the crank arm192 rotates around the center of the rotating shaft 190. The crank lever162 that is attached to a tip of the crank arm 192 by the crank shaft174 moves as writing a track shown by a dotted and dashed line X in FIG.11. The wire 110 that is fixed to the tip of the crank lever 162 movesback and forth correspondingly to a stroke 2S according to length S ofthe crank arm 192, and this displacement movement is imparted to thedriving unit 8 via the wire 110. This displacement movement istransmitted to the lever 70, and makes the lever 70 move like a circulararc.

There is no need to rotate the crank arm 192 sequentially. If the crankarm 192 is displaced by a necessary angle with using a stepper motor ora servo motor, displacement movement of the lever 70 can be obtained aswell.

With the actuator 124, as shown in FIG. 12, a controller 200 thatcontrols the motor 178 is provided. To the controller 200, the pressureapparatus 27, the pressure sensor 29, the motor 178, the encoder 180,the rotation adjustor 182, the power source switch 183, the operationswitch 184, the display 188, etc. are connected. In the controller 200,rotational speed is set to the rotation adjustor 182 by the rotationadjustment dial 186. Rotation of the motor 178 is set to set rotationalspeed. This rotational force is converted to back and forth movement ofthe wire 110 to be imparted to the driving unit 8. Rotation of the motor178 is detected by the encoder 180, difference between rotational speedof the motor 178 and set rotational speed is calculated, and the motor178 is controlled in a predetermined rotational speed. In thisembodiment, by using the controller 200, output pressure P of thepressure apparatus 27 is controlled in accordance with detected pressureof the pressure sensor 29. Another controller may control the pressureapparatus 27, or take in detected pressure of the pressure sensor 29.

E) Cultivation Method

A cultivation method for a cell or tissue by using the culture unit 2will be described with referring to FIGS. 13-16. FIG. 13 is a flowchartshowing processing procedure of cultivation, FIGS. 14A and 14B depictdisposing the cell construct, FIGS. 15A and 15B depict imparting bendingmotion to the cell construct and cancellation thereof, and FIG. 16 is aflowchart showing operation procedure of the actuator.

As shown in FIG. 13, a cultivation process of the cell construct 6includes a preparation (step S1), a cultivation process (step S2) and aposttreatment (step S3). The preparation includes processes of formingthe cell construct 6, a wrapping in a semi-permeable membrane, etc. Thecultivation process includes a bending movement process. In thecultivation process, a curve process (step S21), curve cancellation(step S22), a curve process (step S23) . . . curve cancellation (stepS2N) are repeatedly executed. The posttreatment includes taking out ofthe cell construct 6 whose cultivation is ended from the culture bed 36.

I Preparation (step S1)

As shown in FIGS. 14A and 14B, the cell construct 6 that is a culture isformed. This cell construct 6 is, for example, covered by asemi-permeable membrane.

The cell construct 6 includes one or some of a cell, a cell scaffold, anextracellular matrix generated by the cell and the culture fluid 48.Additives may be included as other elements.

The cell construct 6 may be a three dimensional scaffold where cells aredisseminated and a gel substance, be a three dimensional scaffold wherecells are disseminated being sealed by a semi-permeable membrane, or bea three dimensional scaffold where cells are disseminated and a gelsubstance, both of which is sealed by a semi-permeable membrane. A threedimensional scaffold and a gel substance are composed of a bioabsorbablematerial.

The cell construct 6 also may be a three dimensional scaffold wherecells are disseminated, be a complex of the construct and otherscaffolds, be the above inserted into a bag or a tube of asemi-permeable membrane, be a culture fluid where cells are suspendedbeing enclosed in a bag or a tube of a semi-permeable membrane, or becells and a gel scaffold being enclosed into a bag or a tube of asemi-permeable membrane.

A semi-permeable membrane may be selected out of semi-permeablemembranes whose transmission molecular weight is from 100 (Da) to 1000(kDa), and a selected semi-permeable membrane may be used. An idea ofusing a semi-permeable membrane in cultivation is described inPCT/US2005/027220 (Amorphous cell delivery vehicle treated withphysical/physicochemical stimuli), etc. Since various kinds ofsemi-permeable membrane are provided in accordance with a size of amolecule that can pass through the semi-permeable membrane, themembranes may be used. That is, if a semi-permeable membrane such thatsubstance of a low molecule like nutrition, a necessary gas such asoxygen and waste matters exhausted by a cell in a culture fluid passes,and a cell and a polymeric extracellular matrix are not allowed to passis selected then cells are closed, nutrition and oxygen can be suppliedwhile preventing an outflow of a cell and an extracellular matrix, andeffective cultivation is realized. In this case, as a measure against acase where a semi-permeable membrane prevents passage of nutrition, inthe present invention, bending movement is added. Thus, displacement ofa bending part rises actively, and difference of pressure occurs, thus,move of nutrition is promoted. The cell construct 6 that a blood vesselis still not generated (tissue without a blood vessel) is alsocultivated with bending motion that acts for working a blood vessel anda heart, and simultaneously, the bending motion imparts physicalstimulation to a cell.

The cell construct 6 covered by a semi-permeable membrane is, as shownin FIGS. 14A and 14B, disposed on the disposing part 136 of the culturebed 36. The cell construct 6 is held by the culture bed 36 and disposedthereon.

II Cultivation Process (Step S2)

The cell construct 6 is, as shown in FIGS. 1 and 15A, transferred to theincubator unit 4 that is a culture space along with the culture bed 36.

The culture fluid 48 is supplied into the incubator unit 4, and thecover unit 14 is attached. After the cover unit 4 is attached, foursupporting protrusions 47 that are provided with the cover unit 14lightly press edges of the culture bed 36 and hold the culture bed 36.

If force F is imparted from a back side of the culture bed 36 by thelever 70, as shown in FIG. 15B, the disposing part 136 of the culturebed 36 is curved upwardly by the force F. By this curving, the cellconstruct 6 on the disposing part 136 is also curved. That is, bendingoccurs to the cell construct 6.

If the force F is released from this bending state, the disposing part136 of the culture bed 36 is restored to an original form by itselasticity to become flat. Thus, as shown in FIG. 15A, the cellconstruct 6 on the disposing part 136 shifts into a flat state. In thiscase, on a top face of the cell construct 6, the holding parts 156 and158 of the culture bed 36 exist. The cell construct 6 that is deformedto be convex upwardly is pressed onto the holding parts 156 and 158 inaccordance with restoration of the disposing part 136 to flattendependently on restoration to an original form of the disposing part136.

Such bending movement is repeated (step S21-step S2N), and tissue isformed as necessary cultivation time passes. If the culture unit 2 shownin FIG. 1, etc. is used, a desired pressure P can be acted on the cellconstruct 6 separately from bending by applying the pressure P to thediaphragm 20 of the incubator bottom 12 via the culture fluid 48.

In this cultivation, It is possible to supply the fresh culture fluid 48to the culture space 16 by supplying the culture fluid 48 from the inletport 50 to the culture space 16 to be exhausted from the outlet port 52.The culture fluid 48 is a supply medium that supplies gas such as oxygenand a nutrient, etc. to the cell construct 6, and a transmitting mediumfor transmitting waste matters exhausted from the cell construct 6.

III Posttreatment (Step S3)

The cell construct 6 whose cultivation is completed is taken out fromthe incubator unit 4 (FIG. 1, etc.) with the culture bed 36. The cellconstruct 6 is applied to a restoration region of a human being.

In such process procedure of cultivation, control operation of thecontroller 200 will be described with reference to a flowchart shown inFIG. 16.

If the operation switch 184 is turned on, the display 188 is lighted(step S11), and rotational speed is set in this lighting state (stepS12). By this, rotation is started, and displacement is given from theactuator 124 via the wire 110 to the slider 112 of the driving unit 8(step S13). By back and forth motion of the wire 110, the lever 70 isrotated and the above described movement is imparted to the cellconstruct 6.

If rotation and displacement are generated, set rotational speed andreal rotational speed are displayed on the display 188 (step S14). Bythe controller 200, rotational speed difference between real rotationalspeed and set rotational speed is monitored (step S15). If difference inrotational speeds or uneven rotation reach a predetermined value or overthe predetermined value, it is detected to be anomaly. If anomalyoccurs, an alarm is displayed on the display 188 (step S16).

Second Embodiment

A second embodiment of the present invention will be described withreferring to FIGS. 17 to 19. FIGS. 17 to 19 depict a structural exampleof a cell construct.

Steps of a cultivation method for a cell and/or tissue by using thiscell construct are as follows:

a. Tissue or a cell is taken out from in vivo;

b. The taken tissue is resolved by enzymes and so on and a cell isseparated to select a necessary cell. Every sort of cells can becultivated;

c. If the number of selected cells runs short or, must be grown, thenumber of the cells is once increased by monolayer cultivation and soon;

d. The cell construct is made.

i. In case of an infinite construct (a growth factor or chemist may beadded to one or some of followings if necessary)

-   -   a cell is suspended in a culture fluid    -   a cell is suspended in a hydro-gel    -   a cell is mixed with a gel scaffold        ii. In case of a finite construct (a growth factor or chemist        may be added to one or some of followings if necessary)    -   a cell is suspended in a culture fluid, and the culture fluid is        entered into a cell scaffold such as a collagen sponge and a        chitosan sponge to attach the cell to the scaffold    -   a cell in a sol state is mixed into a scaffold, and the scaffold        is entered in a cell scaffold such as a collagen sponge and a        chitosan sponge to attach the cell to the scaffold and to gel        the cell.        e. A cell construct 6 is configured by entering a cell, etc. in        a tube or a bag of a semi-permeable membrane and sealing the        tube or the bag. A cell and a high molecular are not transmitted        through a semi-permeable membrane, and a low molecular like        nutrition, a chemical and gas such as oxygen in a culture fluid        are transmitted through a semi-permeable membrane.        f. The cell construct 6 is attached to a culture bed 36.        g. If a culture fluid is circulated, a circulation circuit is        prepared for a culture fluid. A gas exchanger is provided with        the circulation circuit, and gas in a cultivation apparatus and        a gas in a culture fluid of the culture circuit can be        exchanged.        h. The cell construct 6 is entered into an incubator unit 4 with        the culture bed 36, and a culture fluid 48 is filled.        i. A cover unit 14 is attached to the incubator unit 4, and the        incubator unit 4 (and the culture circuit) is sealed. The above        are executed in clean environment such as a clean room and clean        bench. After that, since the circuit is sealed, pollution by        germs does not occur even in other places.        j. A driving unit 8 is attached to the incubator unit 4.        k. Temperature and gas concentration is maintained in the best        state.        l. Bending motion is repeatedly imparted to the cell construct        6. A period, size, a motion schedule, etc. of bending motion are        set in advance to operate.        m. If necessary, a culture fluid is circulated and pressure is        applied. Concerning pressure, the best pattern is selected out        of pressure patterns of constant, intermittent, periodical        repeat, etc.        n. After a predetermined time passes, the driving unit is taken        off from the incubator unit 4.

In such cultivation method, the cell construct 6 is, as shown in FIG.17, entered into a tube 202 by a semi-permeable membrane. In this case,the cell construct 6 is constituted of the culture fluid 48 and amixture 204 that gel is mixed with a cell. Openings of both ends of thetube 202 are sealed by a stopper 206.

For example, a pair of cell constructs 6 is attached to the culture bed36, and the above described bending displacement motion is repeatedlyimparted. From this, a cell is propagated, and an extracellular matrixand so on are generated to generate infinite neogenetic tissue.

Seeing this generating process, before cultivation, the cell construct 6is combination of all or a part of a cell, a culture fluid, a hydro geland a gel scaffold. After cultivation, the cell construct 6 is convertedinto combination of all and a part of a cell, a culture fluid, a hydrogel, a gel scaffold, an extracellular matrix or other products of acell.

If a culture extracted from the cell construct 6 is injected into adamaged or deficit part in vivo, original tissue is regenerated at aninjected part. The injected culture amalgamates with tissue therearoundand is integrated.

The cell construct 6 is, as shown in FIG. 18, entered into the tube 202by a semi-permeable membrane. In this case, the cell construct 6 is alsoconstituted of the culture fluid 48 and the mixture 204 that gel ismixed with a cell. Each opening at the edges of the tube 202 is bent,and is sealed by being sandwiched by clips 208. In this case, the tube202 has room. The culture fluid 48 and the mixture 204 are entered sothat a sectional area of the tube 202 that is filled with the culturefluid 48 and a mixture becomes ellipse. These cell constructs 6 areattached to the above described culture bed 36, and repeatedly curved.

By using such cell constructs 6, a cell is propagated, and anextracellular matrix and so on are generated to generate infiniteneogenetic tissue. That is, before cultivation, the cell construct 6 iscombination of all or a part of a cell, a culture fluid, a hydro gel anda gel scaffold. After cultivation, the cell construct 6 is convertedinto combination of all or a part of a cell, a culture fluid, a hydrogel, a gel scaffold, an extracellular matrix and other products of acell.

If such culture is injected into a damaged or deficit part in vivo,original tissue is regenerated at the injected part in vivo, and theinjected culture can amalgamate with tissue therearound.

The cell construct 6 is, as shown in FIG. 19, entered into the tube 202of a semi-permeable membrane. In this case, the cell construct 6 is whata cell is disseminated on a finite cell scaffold 205. Each opening atthe edges of the tube 202 is sealed by the stopper 206.

The single or a plurality of the cell construct 6 are arranged andattached to the culture bed 36, and repeatedly curved. From this, a cellis propagated, and an extracellular matrix and so on are generated togenerate infinite neogenetic tissue. That is, before cultivation, thecell construct 6 is composed of a cell, a cell scaffold such as acollagen sponge and a gel scaffold. After cultivation, the cellconstruct 6 is generated into neogenetic tissue composed of a cell, acell scaffold, an extracellular matrix and other products of a cell.

When a semi-permeable membrane is taken off, inside neogenetic tissue istaken out, and the neogenetic tissue is transplanted to a damaged or adeficit part of a human body by suture, adhesive, etc., an originaltissue is regenerated at a transplanted part and, the neogenetic tissuecan be amalgamated with tissue therearound.

Third Embodiment

A third embodiment the present invention will be described withreferring to FIGS. 20-22. FIG. 20 depicts a cultivation system accordingto a third embodiment, FIG. 21 depicts a structural example of a controlunit and FIG. 22 is a flowchart showing processing procedure of acultivation operation.

In a cultivation system 240, for circulating a culture fluid 48 andsupplying the fresh culture fluid 48 at any time, a culture circuit 244including an incubator unit 4 of a culture unit 2 is provided in aculture room 242. The culture circuit 244 composes a circulation paththat connects a culture fluid tank 248, a gas exchanger 250, a pump 252,a check valve 254, the incubator unit 4 and a pressure adjusting valve256 via a circulation tube 246. In the culture circuit 244, the culturefluid 48 can be circulated optionally. As one aspect of control of thecirculation, for example, if circulation of the culture fluid 48 isstopped or inhibited, the culture circuit 244 may be shut. The abovepump 252 is composed of a piston pump, a syringe pump and a peristalticpump, etc. With the circulation tube 246, for example, a flow sensor 268that detects a flow of the culture fluid 48 by a method such as countingdrops of the culture fluid 48 is provided.

In the culture room 242, a temperature adjustor 258, a gas concentrationadjustor 260 and a controller 262 are provided. In the culture room 242,temperature necessary for cultivation is set by the temperature adjustor258, constant gas concentration is maintained by the gas concentrationadjustor 260, and circulation of the culture fluid 48 and pressure inthe incubator unit 4 are controlled by the controller 262.

To the incubator unit 4, bending stress is imparted from an actuator 124via a cable 119. In this case, the actuator 124 is provided in theculture room 242. The actuator 124 may be provided outside the cultureroom 242.

Motion of the actuator 124 may be controlled along with pressurizedcirculation of the culture fluid 48, or controlled independently. Inthis case, pressure application to the culture fluid 48 and bendingstress by the actuator 124 are imparted to the cell construct 6.

For the cell construct 6, to control imparting of bending displacement,circulation of the culture fluid 48 and pressure in the incubator unit 4together, as shown in FIG. 21, a control system may be configured. Inthe controller 262, rotational speed of a motor 178 and the amount ofcirculation of the culture fluid 48 are set from an input apparatus 264,and the motor 178 and the pump 252 are driven. The controller 262 isconfigured by a computer including a CPU (Central Processing Unit), aROM (Read-Only Memory) and a RAM (Random-Access Memory).

The motor 178, an encoder 180 and the controller 262 executedisplacement imparting control. Rotation of the motor 178 is detected bythe encoder 180, and detected information is inputted to the controller262.

The pressure adjusting valve 256, the pump 252, the flow sensor 268 andthe controller 262 execute culture fluid transmission control. In thisculture fluid transmission control, if rotational speed of the pump 252rises, the amount of a flow of the culture fluid 48 increases. Pressureby fluid transmission can be adjusted by the pressure adjusting valve256. A flow of the culture fluid 48 is, for example, detected by theflow sensor 268 with using a method such as counting drops of theculture fluid 48, and detected information is used in controlinformation such as pump control.

The pressure apparatus 27, the pressure sensor 29 and the controller 262execute pressure application control. The pressure sensor 29 detectspressure, and by detected information, pressure application control isexecuted.

A temperature sensor 280 and a heater 282 that constitute thetemperature adjuster 258, and the controller 262 execute temperatureadjustment control. Based on detected information of the temperaturesensor 280, heat generation temperature of the heater 282 is controlled.A CO₂ concentration censor 284, a CO₂ electromagnetic valve 286, an O₂concentration sensor 288, and an N₂ electromagnetic valve 290 thatconstitute the gas concentration adjuster 260, and the controller 262execute gas concentration control. Based on detected information of theCO₂ concentration sensor 284, a degree of opening of the CO₂electromagnetic valve 286 is controlled. Based on detected informationof the O₂ concentration sensor 288, a degree of opening of the N₂electromagnetic valve 290 is controlled. This control of temperatureadjustment and gas concentration execute environment control in theculture room 242. That is, according to gas concentration atmosphere inthe culture room 242, in the gas exchanger 250, gas is exchanged betweenthe culture fluid 48 and the culture room 242.

In such cultivation system 240, as shown in FIG. 22, turning on a powersource switch 292 (step S51) lights a display 266 (step S52). Thus,after input of a value of environment control (step S53) and input of avalue of control operation (step S54), setting values are displayed onthe display 266 (step S55). Here, an operation switch 294 is turned on(step S56).

If the cultivation system 240 is in an operation state, temperatureadjustment (step S57), gas concentration adjustment (step S58), pressureapplication control (step S59), culture fluid transmission drive (stepS60) and displacement imparting drive (step S61) are executed, and adriving state is displayed on the display 266 (step S62).

A driving state necessary for displacement imparting is monitored (stepS63). If there is anomaly, after an alarm is displayed, drive is stopped(step S64), the cultivation system 240 is reset (step S65), a cause ofanomaly is taken off (step S66), steps S51-S63 are operated again, andcultivation is ended (step S67). After ending cultivation, the operationswitch 294 is turned off (step S68), and a cultivation process is ended(step S69).

Fourth Embodiment

A fourth embodiment of the present invention will be described withreferring to FIGS. 23 and 24. FIG. 23 depicts a culture unit accordingto a fourth embodiment, and FIG. 24 depicts a section of FIG. 23 alongan XXIV-XXIV line. In FIGS. 23 and 24, the same parts as those in FIGS.1-8 are denoted by the same reference numerals.

In the first to third embodiments, a process of imparting bendingdisplacement to the cell construct 6 is described. The present inventioncan apply to an extension and contracting process of a cell construct600. A desired displacement can be imparted to the cell construct 600without generating change of inner pressure in a culture space 16 of anincubator unit 4 by movement of a lever 70.

In the incubator unit 4 of this embodiment, the rectangular culturespace 16 is formed. One end of the cell construct 600 is attached to aprotrusion 296 that protrudes from a shelf 295 of the culture space 16,and a tip of the lever 70 is rotatably fixed to a holding frame 298 thatis attached to the other end of the cell construct 600. In thisembodiment, a fixing pin 300 is protruded over the holding frame 298. Anengaging ring 302 that is formed at the tip of the lever 70 is movablyengaged with this fixing pin 300.

From such structure, if the lever 70 is rotated in a circular arc bystress imparted from an actuator 124 (FIGS. 8 and 9) to a driving unit 8via a wire 110, the cell construct 6 is extended and contracted indirections of arrows m and n in accordance with rotation in circulatearc, and stimulation, imparting of tension and cancellation thereofnecessary for cultivation can be repeatedly executed.

In this case, a culture fluid 48 is poured into the culture space 16from an inlet port 50, poured out from an outlet port 52, thus, thefresh culture fluid 48 can be supplied to the cell construct 600.

Features Extracted from the Embodiments

Concerning features extracted from the embodiments described above,features except matters described in claims are as follows. Listedfeatures do not limit the present invention.

(1) The above cultivation apparatus, wherein the bed and the lever aredisposed in parallel or perpendicularly.

(2) The above cultivation apparatus, wherein the incubator unit issealed structure.

(3) The above cultivation apparatus, wherein an edge of the lever has anaction unit.

(4) The above cultivation apparatus, wherein the bed holds the culture,curves by receiving pressure from the lever and restores a state beforeapplying pressure by cancelling the pressure application.

(5) The above cultivation apparatus, wherein the bed comprises a holdingunit, at an edge of the bed, that the incubator unit holds, and areceiving unit, at a middle part of the bed, that receives action fromthe lever.

(6) The above cultivation apparatus, wherein the lever comprises aflange that has circular arc face in the vicinity of the fulcrum, ajoint part having a circular arc face correspondingly to the flange isincluded in the incubator unit, and the circular arc face of the flangeand the circular arc face of the joint part are slidably contacted.

(7) The above cultivation apparatus, wherein in the joint part, when thelever is rotated, the center of rotation of the lever corresponds with acenter of a sealing part of the lever.

(8) The above cultivation apparatus, wherein a driving unit that impartsdriving force to a rear end of the lever extracted from the incubatorunit is provided at a side of the incubator unit.

(9) The above cultivation apparatus, wherein the driving unit isdisposed detachably and attachably in the incubator unit.

(10) The above cultivation apparatus, comprising a control part thatcontrols the pressure application imparted from the driving unit to thelever periodically and consequently, and/or controls applicationpressure velocity.

(11) The above cultivation apparatus, wherein to the incubator unit, aculture fluid is supplied from a supplying port and exhausted from anexhausting port.

(12) A cultivation apparatus comprising a diaphragm in an incubatorunit, wherein pressure can be applied into the incubator unit via thediaphragm.

(13) The above cultivation apparatus, wherein the driving unit comprisesa housing unit that is disposed at a side of the incubator unit,

a first slider that is slidably held in the housing, slides by drivingforce applied from an outside, and generates circular arc movement tothe lever, and

a second slider that is slidably held in the housing, and acts restoringforce on the lever in an opposite direction from the slider.

(14) The above cultivation apparatus, wherein the housing comprises aninserting port where the lever is inserted, and

a guide part that guides an end of the lever inserted from the insertingport between the first slider and the second slider.

Result of Experiment

A result of an experiment using the above cultivation apparatus and thecultivation system will be described with referring to FIGS. 25 to 29.

FIG. 25 shows a cell construct. This cell construct is composed ofentering a cell suspended in a culture fluid into a tube of asemi-permeable membrane. As shown in FIG. 26, the cell construct isfixed to a culture bed, and is accommodated in an incubator unit(chamber). In this case, a driving unit is separated from the incubatorunit.

Pressure from an actuator acts on a culture unit, and bending motion isimparted to the cell construct. The actuator is disposed outside aculture room. A cable was penetrated through a door of the culture roomto be connected to the driving unit. An operation state of the actuatorcould be confirmed by a display.

The actuator converts a rotational movement of a motor to linearmovement by a crank. By selection of the length of a crank arm, width ofback and forth of a wire could be adjusted, and in accordance with thisselection, a size of bending imparted to the cell construct can beadjusted.

In this experiment, pressure application operation is limited to bendingmotion, atmospheric pressure is maintained and the culture fluid iscirculated. Pressure and bending motion by the actuator are impartedindividually, irrelevantly and solely. In the experiment, for example,it can be considered that pressure equal to or over 0.5 (MPa) isimparted.

FIGS. 27 to 29 show an experiment of vertebrae organ cultivation of atwo days old mouse. In the experiment, a vertebra taken out from the twodays old mouse is disposed on the culture bed (FIG. 27) and bendingmotion of 0.1 (Hz) frequencies is imparted to be cultivated for tendays. In this experiment, no pressure is applied.

As a comparison example, static cultivation is executed. FIGS. 28 and 29show static cultivation for ten days. After ten days, a section of anorgan is toluidine blue-stained, and condition of a cell existence isobserved. In Figs., a stained part can not be expressed. A part wherebrightness falls down (a stained part) shows existence of a living cell.In the static cultivation, cell density inside discs does not rise, anddisplacement of matrixes can be seen (a of FIG. 28).

On the contrary, in vertebrae where bending motion and displacement areimparted, growth of cells and store of neogenetic matrixes inside discscan be seen. (b of FIG. 29).

From the result of the experiment, in the cultivation of impartingbending motion, growth of cells and store of neogenetic matrixes can beseen as prepared with the static cultivation. Thus, it can be guessedthat the bending motion gives stimulation to the cell construct, andmakes substance migration promote.

While the present invention has been described with the preferredembodiments, the description is not intended to limit the presentinvention.

The present invention relates to a cultivation apparatus for a cultureincluding a cell and/or tissue. A cultivation apparatus for a celland/or tissue proper to a region of a human body, etc. is provided. Byimparting bending motion to a cell construct, tissue cultivation ofvertebra, etc. can be executed efficiently. So, the present invention isuseful.

All examples and conditional language recited herein are intended forpedagogical purposes to aid the reader in understanding the inventionand the concepts contributed by the inventor to furthering the art, andare to be construed as being without limitation to such specificallyrecited examples and conditions, nor does the organization of suchexamples in the specification relate to a showing of the superiority andinferiority of the invention. Although the embodiment(s) of the presentinvention(s) has(have) been described in detail, it should be understoodthat the various changes, substitutions, and alterations could be madehereto without departing from the spirit and scope of the invention.

The invention claimed is:
 1. A cultivation apparatus for a cultureincluding a cell and/or tissue, comprising: a bed that includes adisposing part which is formed by an elastic member having elasticity,the culture being disposed on the disposing part; an incubator unit thataccommodates the bed, which includes the disposing part on which theculture is disposed, the bed being fixed to the incubator unit; a leverthat penetrates from an inside of the incubator unit to an outsidethereof, and can move in a circular arc around a fulcrum as a center,the fulcrum being set at a wall of the incubator unit or in the vicinitythereof, the lever having an action unit at one edge thereof, the actionunit being disposed in the incubator unit; and a driving unit thatimparts driving force to another edge of the lever and releasesimpartation of the driving force, the driving unit making the actionunit of the lever move in a circular arc to push a back side of thedisposing part to curve the disposing part when imparting the drivingforce to the lever, wherein the disposing part of the bed is curved bythe impartation of the driving force to the lever, and the elasticity ofthe disposing part restores the disposing part to an original form withrelease of the impartation of the driving force, to deform the culturein extension and contraction.
 2. A cultivation apparatus according toclaim 1, wherein the bed and the lever are disposed in parallel orperpendicularly.
 3. A cultivation apparatus according to claim 1,wherein the incubator unit is a sealed structure.
 4. A cultivationapparatus according to claim 1, wherein the bed holds the culture,curves by receiving pressure from the lever and restores a state beforeapplying pressure by cancelling the pressure application.
 5. Acultivation apparatus according to claim 1, wherein the bed comprises aholding unit, at the edges of the bed, that the incubator unit holds;and a receiving unit, at a middle part of the bed, that receives actionfrom the lever.
 6. A cultivation apparatus according to claim 1, whereinthe lever comprises a flange having a circular arc face in the vicinityof the fulcrum, a joint part having a circular arc face correspondinglyto the flange is included in the incubator unit, and the circular arcface of the flange and the circular arc face of the joint part areslidably contacted.
 7. A cultivation apparatus according to claim 6,wherein in the joint part, when the lever is rotated, the center ofrotation of the lever corresponds with a center of a sealing part of thelever.
 8. A cultivation apparatus according to claim 1, wherein thedriving unit is provided at a side of the incubator unit, and impartsdriving force to a rear end of the lever extracted from the incubatorunit.
 9. A cultivation apparatus according to claim 1, wherein thedriving unit is disposed detachably and attachably to the incubatorunit.
 10. A cultivation apparatus according to claim 1, furthercomprising: a control part that controls the driving force imparted fromthe driving unit to the lever periodically and consequently, and/orcontrols application pressure velocity.
 11. A cultivation apparatusaccording to claim 1, wherein a culture fluid is supplied to theincubator unit from a supplying port and exhausted from an exhaustingport.
 12. A cultivation apparatus according to claim 1, furthercomprising: a diaphragm that is disposed over a window, the window beingformed in the incubator unit, wherein pressure can be applied into theincubator unit via the diaphragm.
 13. A cultivation apparatus accordingto claim 1, wherein the driving unit comprises a housing unit that isdisposed at a side of the incubator unit; a first slider that isslidably attached to the housing, slides by driving force applied froman outside, and generates circular arc movement to the lever; and asecond slider that is slidably attached to the housing, and actsrestoring force on the lever in an opposite direction from the slider.14. A cultivation apparatus according to claim 13, wherein the housingcomprises an inserting opening where the lever is inserted; and a guidepart that guides an end of the lever, which is inserted through theinserting opening, to a position between the first slider and the secondslider.